Voltage-gated calcium channels are a heterogeneous family of membrane proteins, which respond to depolarization by opening a calcium-selective pore through the plasma membrane. The influx of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression. In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Calcium entry further affects neuronal function by directly regulating calcium-dependent ion channels and modulating the activity of enzymes such as protein kinase C and calcium-dependent calmodulin-dependent protein kinase II. Furthermore, an increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitters. Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and is implicated in long-term changes in neuronal activity. In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. Recently, mutations identified in human and mouse calcium channel genes were found to account for several disorders including, familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. (See, for example, Fletcher, C. F., et al., Cell (1996) 87:607-617; Burgess, D. L., et al., Cell (1997) 88:385-392; Ophoff, R. A., et al., Cell (1996) 87:543-552; Zhuchenko, O., et al., Nature Genetics (1997) 15:62-69. The clinical treatment of some disorders has been aided by the development of therapeutic calcium channel modulators or blockers. Janis, R. J. and Triggle, D. J. (1991) in Calcium Channels: Their Properties Functions Regulation and Clinical Relevance, CRC Press, London).
Native calcium channels are classified by their electrophysiological and pharmacological properties as either high voltage-activated (L, N, P, and Q types) or low voltage-activated channels (T-type). R-type channels have biophysical properties similar to both high and low voltage-activated channels. (For reviews see McCleskey, E. W. and Schroeder, J. E., Curr. Topics Membr. (1991) 39:295-326, and Dunlap, K., et al., Trends Neurosci. (1995) 18:89-98.) T-type channels are a broad class of molecules that transiently activate at negative potentials and are highly sensitive to changes in resting potential. The L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties among the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them. L-type channels are sensitive to dihydropyridine (DHP) blockers, N-type channels are blocked by the Conus geographus peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta. A fourth type of high voltage-activated Ca2+ channel (Q-type) has been described, although whether the Q- and P-type channels are distinct molecular entities is controversial. Conductance measurements of several types of calcium channels have not always fallen neatly into any of the above classes and there is variability of properties even within a class, suggesting that additional calcium channels subtypes remain to be classified.
Biochemical analyses show that neuronal calcium channels are heterooligomeric complexes consisting of three distinct subunits (α1, α2δ and β) (reviewed by De Waard, M., et al., in Ion Channels, Volume 4, (1997) edited by Narahashi, T., Plenum Press, New York). The α1 subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel blockers. The mainly extracellular α2 is disulphide-linked to the transmembrane δ subunit and both are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the α1 subunit. A fourth subunit, γ, is unique to L-type calcium channels expressed in skeletal muscle T-tubules. The isolation and characterization of γ-subunit-encoding cDNA's is described in U.S. Pat. No. 5,386,025, which is incorporated herein by reference.
The DNA's encoding the amino acid sequences of seven different types of α1 subunits (α1A, α1B, α1C, α1D, α1E, α1F and α1S) and four types of β subunits (β1, β2, β3 and β4) have been cloned. (Reviewed in Stea, A., et al., “Voltage-gated calcium channels” in Handbook of Receptors and Channels (1994) Edited by R. A. North, CRC Press). PCT Patent Publication WO 95/04144, which is incorporated herein by reference, discloses the sequence and expression of α1E calcium channel subunits.
In some expression systems the α1 subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits. Until recently, the reported modulatory affects of β subunit coexpression were to mainly alter kinetic and voltage-dependent properties. It has now been shown that β subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, E., et al., EMBO J. (1994) 13:5032-5039; Stea, A., et al., Neuron (1995) 15:929-940; Bourinet, E., et al., Proc. Natl. Acad. Sci. (USA) (1996) 93:1486-1491.)
The α2δ subunits comprise at least four types encoded by different genes. The first subunit identified was α2δ-1 from rabbit skeletal muscle (Ellis, et al., Science (1988) 241:1661-1664). Five tissue-specific splice variants exist (Angelotti, T. and Hofmann, F., FEBS Lett. (1996) 397:331-337). α2δ-2, -3 and -4 have been identified recently in human and mouse (Klugbauer, N., et al., J. Neuroscience (1999) 19:684-691; Qin, N., et al., Mol. Pharmacol. (2002) 62:485-496). These α2δ subunits share 30% to 56% amino acid identity with the α2δ-1 subunit as well as several structural motifs, such as similar hydrophobicity profiles, glycosylation sites and cysteine residues. α2δ-1 and α2δ-2 subunits are expressed in many tissues including the brain and heart, while α2δ-3 is found only in the brain (Klugbauer, et al., 1999 (supra)). A recent report showed that IGF-1 stimulates α2δ-3 expression in cultured rat atrial myocytes. (Chu, P.-J., J. Mol. Cell. Cardiology (2003) 35:207-215.) The α2δ-4 subunit is distributed in certain cell types of the pituitary, adrenal gland, colon and fetal liver (Qin, et al., 2002 (supra)).
A number of physiological roles have been proposed for the α2δ-2 subunit, including acting as a tumor suppressor gene, and a mutation in the mouse homolog, resulting in a truncated α2δ-2 has been identified as a contributing factor to the ducky epileptic phenotype (Gao, B., et al., J. Biol. Chem. (2000)275:12237-12242; Brodbeck, J., et al., J. Biol. Chem. (2002) 277:7684-7693). The antiepileptic gabapentin binds to the α2δ-1 and -2 subunits, but not to α2δ-3 (Marais, E., et al., Molec. Pharmacol. (2001) 59:1243-1248).
α2δ-1 increases the current density of calcium channels by increasing the amount of functional channel at the cell surface and enhances dihydropyridine binding to L-type channels and ω-conotoxin GVIA to N-type channels (Brust, P. F., et al., Neuropharmacology (1993) 32:1089-1102; Felix, R., et al., J. Neurosci. (1997) 17:6884-6891). α2δ-2 and α2δ-3 significantly enhance and modulate the Ca2+ current through a number of HVA and LVA channels (Klugbauer, et al. (1999) (supra); Gao, et al. (2000) (supra); Hobom, M., et al., Eur. J. Neurosci. (2000) 12:1217-1226).
Recently, the molecular cloning of α2δ-2 and α2δ-3 subunits from rat atria was reported. (Chu, P-. J., et al., 2003 (supra)). Cloning of rat α2δ-2 and α2δ-3 subunits from rat brain tissue has not been previously disclosed.